Synthesis of Azo Bonded Immunoregulatory Compounds

ABSTRACT

Methods are disclosed for preparing compounds of Formula I: where R 1 , R 3 , and R 4 are independently hydrogen or C 1  to C 4  alkyl, and R 2  is: where R 5  is selected from the group consisting of hydrogen and C 1  to C 4  alkyl, or where R 6 , R 7  and R 8  are independently hydrogen or C 1  to C 4  alkyl; or the esters or pharmacologically acceptable salts thereof. The methods can involve converting a suitably functionalized aniline compound to a diazonium salt (which aniline compound can be first formed by reduction of a nitrobenzene) and coupling the diazonium salt with a suitably functionalized benzene compound. The suitably functionalized aniline compound either includes a primary alcohol or aldehyde group, which is then oxidized to a carboxylic acid group, or includes a nitrile or amide group, which is hydrolyzed to a carboxylic acid group. The methods can also involve the direct coupling (via reduction of nitro groups to form an azo linkage) of suitably functionalized nitrobenzenes. The compounds and or their metabolites can be used to treat or prevent various diseases, particularly inflammatory conditions of the GI tract.

1. FIELD OF THE INVENTION

The present invention relates to immunoregulatory compounds, methods of producing same and methods of treating diseases therewith.

2. BACKGROUND OF THE INVENTION

Many people suffer from inflammatory bowel disease (IBD). IBD is a generic term used to refer to two inflammatory diseases, ulcerative colitis and Crohn's disease, and various medications are being used to treat inflammatory bowel disease. For example, mesalamine, 5-aminosalicylic acid (5-ASA) is used to treat ulcerative colitis, but is associated with various side effects. It can be absorbed as it passes through the GI tract, which can adversely affect the amount of mesalamine that reaches the lower GI tract, particularly the colon and rectum. Sulfasalazine has also been used, but is metabolized in the body to form mesalamine (5-aminosalicylic acid (5-ASA)) and sulfapyridine. Accordingly, sulfasalazine is associated with several adverse side affects, including nausea, vomiting, abdominal discomfort, male infertility, and headache. These adverse side effects are usually attributed to the activity of sulfapyridine in the GI tract, as well as that absorbed into the system. Olsalazine has also been used to treat ulcerative colitis, but is both relatively expensive to make and associated with adverse side effects including diarrhea.

Efforts have been made to minimize the side effects associated with these compounds, including efforts by the present inventors to provide compounds that, when reaching the gut mucosa, break down into one or more active compounds useful for treating inflammatory bowel disorders. Examples of such compounds are described, for example, in U.S. Pat. No. 6,583,128 to Ekwuribe et al.

It would be beneficial to provide additional synthetic methods for preparing these compounds. The present invention provides such synthetic methods.

3. SUMMARY OF THE INVENTION

Synthetic methods for preparing compounds of the following formula are provided:

where R¹, R³, and R⁴ are independently hydrogen or C₁ to C₄ alkyl, and R² is:

where R⁵ is selected from the group consisting of hydrogen and C₁ to C₄ alkyl, or

where R⁶, R⁷ and R⁸ are independently hydrogen or C₁ to C₄ alkyl, as well as the esters or pharmaceutically acceptable salts of such compounds.

The compounds prepared by these methods can be included in pharmaceutical compositions, and used in methods of treating inflammatory conditions.

The active pharmaceutical ingredient may further comprise a compound of Formula III:

The first step of the synthesis typically starts with an aniline compound, which is converted to a diazonium salt. In one embodiment, the aniline compound to be converted to the diazonium salt includes a primary alcohol or aldehyde group, which can be oxidized to a carboxylic acid group following formation of the diazonium salt and coupling to a desired molecule.

In another embodiment, the aniline compound to be converted to the diazonium salt includes a nitrile or amide group, which can be hydrolyzed to the carboxylic acid following formation of the diazonium salt and coupling to a desired molecule.

The second step of the synthesis involves forming a diazonium salt, which can be performed using known chemistry. The resulting diazonium salt is then coupled with a compound of the formula:

This coupling step typically provides predominantly a substitution at the 5-position of the salicylate, or a para substitution. In the embodiments where the aniline includes a primary alcohol or aldehyde group, the next step involves oxidizing the primary alcohol or aldehyde group to a carboxylic acid group. This can be accomplished using traditional oxidation chemistry, but optionally is performed using a catalytic amount of a chromium (VI) oxidant or other suitable oxidant in the presence of a stoichiometric amount of periodic acid.

In the embodiments where the aniline includes a nitrile or amide group, the next step involves hydrolysis of the amide or nitrile to the carboxylic acid. The hydrolysis can be either acid or base catalyzed. However, nitrilases and/or amidases can also be used, which typically results in milder reaction conditions.

In either embodiment, following the oxidation of the primary alcohol or aldehyde to the carboxylic acid, or hydrolysis of the amide or nitrile to the carboxylic acid, a pharmaceutically acceptable carboxylate salt or suitable ester (i.e., where R¹ on the CO₂R¹ group is alkyl) can be formed using routine chemistry.

In an alternative embodiment of the first approach (where the aniline to be converted to the diazonium salt includes a primary alcohol or aldehyde group, the methods first involve converting a compound of the formula:

to a compound of formula:

This conversion can be performed using standard reaction conditions well known to those of skill in the art, for example, as shown below with respect to the nitrobenzene including the primary alcohol group:

In an alternative embodiment of the second approach (where the aniline to be converted to the diazonium salt includes a nitrile or amide group), the methods first involve converting a compound of the formulas:

to a compound of the formulas:

The amide group can optionally include one or two alkyl, aryl, arylalkyl or alkylaryl groups in place of one or both of the hydrogen atoms, which groups are removed when the amide is hydrolyzed to the carboxylic acid. However, since such groups would not be present in the final molecule, it is often easier to simply use an unsubstituted amide group (i.e., CONH).

This conversion can be performed using standard reaction conditions well known to those of skill in the art, for example, as shown below:

In still another embodiment, the starting materials for the reaction are nitrobenzene compounds such as:

which are coupled with a compound of the formula:

using a reagent such as lithium aluminum hydride (“LiAlH₄”), which is known in the art to couple nitrobenzenes to form azo compounds. Desired compounds are isolated and purified.

Alternatively, two compounds of the formula:

can be coupled directly, forming an azo linkage. Potential limitations to this approach are that when two different starting nitrobenzenes are used, three types of azo couplings are possible (A-A, A-B, B-B). Further, the reducing agent can reduce aldehyde, amide and nitrile groups, which then need to be reoxidized to form the carboxylic acid moieties. For this reason, it can be advantageous to perform the direct azo coupling of nitrobenzenes with starting materials that already include a carboxylic acid moiety (or carboxylate salt form thereof).

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates embodiments of the synthesis routes described herein.

FIG. 2 illustrates additional embodiments of the synthesis routes described herein.

5. DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described with respect to preferred embodiments described herein. It should be appreciated however that these embodiments are for the purpose of illustrating the invention, and are not to be construed as limiting the scope of the invention as defined by the claims.

As shown below, various synthetic methods are provided for preparing the compounds described herein.

In a first embodiment (Scheme I in FIG. 1), an aniline derivative including a primary alcohol or aldehyde group is used, where the primary alcohol or aldehyde group is ultimately oxidized to form a carboxylic acid. The carboxylic acid group can optionally be converted to a pharmaceutically acceptable carboxylate salt or ester.

In a second embodiment (Scheme II in FIG. 2), an aniline derivative including a nitrile or amide group is used, where the nitrile or amide group is ultimately hydrolyzed to form a carboxylic acid. The first step in either embodiment involves forming a diazonium salt.

In a third embodiment, nitrobenzenes (rather than anilines) are used as starting materials, and are reduced to form the aniline starting materials used in the first two embodiments.

In a fourth embodiment, two nitrobenzenes are directly coupled to form an azo linkage, using a reducing agent such as lithium aluminum hydride.

Each of these reaction conditions and the associated starting materials will be better understood with reference to the following detailed description.

5.1 The Compounds

The compounds synthesized using the methods described herein include those having the following formula:

where R¹, R³, and R⁴ are independently hydrogen or C₁ to C₄ alkyl. Preferably, R¹, R³, and R⁴ are independently selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂. More preferably, R¹, R³, and R⁴ are H or CH₃. R² is:

R⁵ is selected from the group consisting of hydrogen and C₁ to C₄ alkyl. Preferably, R⁵ is selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂. More preferably, R⁵ is H or CH₃ and, most preferably, R⁵ is H.

R⁶, R⁷ and R⁸ are independently hydrogen or C₁ to C₄ alkyl. Preferably, R⁶, R⁷ and R⁸ are independently selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂. More preferably, R⁶, R⁷ and R⁸ are independently H or CH₃.

5.2 Synthetic Methods

The individual reaction steps are discussed in more detail below.

5.2.1 Nitrobenzene Reduction to Aniline Starting Materials

In an alternative embodiment of the embodiment shown in Scheme 1 (where the aniline to be converted to the diazonium salt includes a primary alcohol or aldehyde group), the methods first involve converting a compound of the formula:

to a compound of formula:

This conversion can be performed using standard reaction conditions well known to those of skill in the art, for example, as shown below:

In an alternative embodiment of the embodiment in Scheme II (where the aniline to be converted to the diazonium salt includes a nitrile or amide group), the methods first involve converting a compound of the formulas:

to a compound of the formulas:

where R₁ is as defined above. The amide group can include one or two alkyl, aryl, arylalkyl or alkylaryl groups (in addition to those defined by R¹) such as are well known in the art in place of one or both of the hydrogen atoms, which groups are removed when the amide is hydrolyzed to the carboxylic acid. However, since such groups would not be present in the final molecule, it is often easier to simply use an unsubstituted amide group (i.e., CONH₂).

This conversion can be performed using standard reaction conditions well known to those of skill in the art, for example, as shown below:

5.2.2 Conversion of Anilines to Diazonium Salts

The first step in Scheme I involves converting an aniline compound to a diazonium salt. The aniline compounds include a primary alcohol or aldehyde group, and the conversion involves the known reaction with NaNO₂ to convert anilines to diazonium salts. Such reaction conditions are well known to those of skill in the art, and the reactions are shown below.

The first step in Scheme II also involves converting an aniline compound to a diazonium salt. The aniline compounds include a nitrile or amide group, and the conversion similarly involves the known reaction with NaNO₂ to convert anilines to diazonium salts. The reactions are shown below.

In the reactions shown above, R¹ is as defined elsewhere in this application. It is also contemplated that with respect to the amide groups, any substitution on the amide linkage that does not interfere with the subsequent chemistry is acceptable, since the amide is later hydrolyzed to the carboxylic acid and such groups will not appear, in any case, in the final molecule. Such groups include, for example, C₁₋₁₀ straight, branched or cyclic alkyl groups, aryl groups such as those including one or more benzene or naphthalene rings, alkyl groups substituted with aryl groups (alkylaryl groups), aryl groups substituted with one to five alkyl groups (arylalkyl groups) and the like.

5.2.3 Electrophilic Aromatic Substitution Using Diazonium Salts

Electrophilic aromatic substitution reactions using diazonium salts are well known. The following two aromatic compounds are substituted with the previously described diazonium salts:

With respect to the first compound, substitution primarily takes place at a position para to the phenol group and meta to the carboxylic acid group. With respect to the second compound, substitution primarily takes place at a position para to the substituted (with R¹) acetic acid group. The following reactions take place in connection with Scheme I.

The following reactions take place with respect to Scheme II:

5.2.4 Oxidation of Primary Alcohol and Aldehyde Groups

With respect to Scheme I, the primary alcohol or aldehyde groups in the intermediate resulting from the diazonium coupling reaction are then oxidized to carboxylic acid groups. The reactions are shown below:

In some embodiments, particularly those embodiments where there is a phenol group present, the oxidation chemistry may oxidize the phenol to a quinone-type compound. In such embodiments, the phenol can be protected with conventional protecting group chemistry (e.g., as a silyl ether, a THP ether, a benzyloxy, an ester, and the like), and deprotected after the oxidation step. Examples of suitable protecting groups for phenol groups which are amenable to various oxidation conditions are well known to those of skill in the art ((see Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991, the contents of which are hereby incorporated by reference).

U.S. Pat. No. 6,150,554 to Li et al., the contents of which are hereby incorporated by reference, teaches the oxidation of primary alcohols to carboxylic acids using catalytic CrO₃ and periodic acid (referred to in the literature as both HIO₄ and H₅IO₆) as the stoichiometric oxidant. This chromium catalyzed oxidation method avoids the chromium disposal issues associated with running a typical Jones oxidation reaction with stoichiometric CrO₃, reduces the epimerization of any α-chiral centers, and is a one step procedure. The reaction is mild, rapid, high yielding and only requires 1-2 mol % of CrO₃.

The oxidation of primary alcohols to carboxylic acids is well known in the art of organic synthesis. Typical reagents include chromium (VI) oxidizing agents (including, but not limited to, sodium and potassium dichromate, chromic acid, and the like), and manganese (VII) oxidizing agents such as potassium permanganate. The oxidation of aldehydes to carboxylic acids is also well known. While the same strong oxidizing agents used to oxidize primary alcohols can be used, much milder oxidizing agents can also be used. For example, ozone (or oxygen) and ultraviolet light and/or a suitable catalyst can often be used. Representative reaction conditions are described, for example, in U.S. Pat. No. 6,680,395 to Springer, the contents of which are hereby incorporated by reference.

Industrially, it is often preferred to use the mildest and/or least expensive oxidation conditions that result in the desired products. Accordingly, it can be preferred to use oxygen or ozone as an oxidizing agent where appropriate, or to use catalytic chromium (VI) salts along with a co-oxidizing agent such as periodic acid to regenerate the catalytic chromium (VI) salts where appropriate.

5.2.5 Hydrolysis of Nitrile and Amide Groups

The embodiment in Scheme II does not require the use of primary alcohols in the starting material, so that oxidation from a primary alcohol to a carboxylic acid group can be avoided. This embodiment involves the use of an aniline that includes a nitrile (—CN) or amide (—C(O)NR¹ ₂) group in place of the primary alcohol group. The nitrile or amide groups can be hydrolyzed to a carboxylic acid group after the diazonium salt is coupled to a desired aromatic ring. The reactions are shown below.

Reaction conditions for converting a nitrile to a carboxylic acid group are well known to those of skill in the art, and are described, for example, in March, J. Advanced Organic Chemistry; 3rd ed., John Wiley: New York, (1985) and House, Modern Synthetic Reactions, 2d. ed., W. A. Benjamin, Inc., Menlo Park, Calif., (1972). The conditions involve either using an acid or a base to catalyze the hydrolysis. A tautomer of an amide is believed to be an intermediate in the hydrolysis of a nitrile group to a carboxylic acid. For this reason, an amide group can be used in place of the nitrile for purposes of carrying out the synthetic method.

Biocatalytic hydrolysis of nitrites to carboxylic acids can be carried out under mild conditions, without affecting other functional groups. The nitrile group thus functions as a “masked” acid group. Nitrilase enzymes (for example, those from Arabidopsis thaliana, A/caligenesfaecalis, Pseudomonas fluorescens, Rhodococcus rhodochrous, and Rhodococcus sp.) are available commercially from companies such as Sigma-Adrich. The chemistry is described, for example, in Effenger and Osswald, “Catalyst for the (E)-selective hydrolysis of (E,Z)-α,β-unsaturated nitrites to carboxylic acids,” Tetrahedron: Asymmetry, 12,:2581 (2001); Effenger and Osswald, “Selective hydrolysis of aliphatic dinitriles to monocarboxylic acids, Synthesis, 1866 (2001); and Effenger and Osswald, “Enantioselective hydrolysis of (1)-arylacetonitriles, Tetrahedron: Asymmetry, 12:279 (2001), the contents of which are hereby incorporated by reference. Because the biocatalytic hydrolysis can involve milder conditions than either the acid or basic hydrolysis of nitriles, this method can be preferred, particularly for industrial synthesis of the compounds described herein.

A large number of amidases (enzymes that cleave amide groups) are also known and can be used to hydrolyze the amides to carboxylic acids. One potential advantage of using the amidases is that they tend to be selective for one enantiomer over another. In some embodiments, the compounds are formed as racemic mixtures or mixtures of diastereomers, and the amidases allow (via enzymatic resolution) the individual enantiomers or diastereomers to be separated. Of course, conventional methods, such as selective crystallization, chiral chromatography and the like, as are well known to those of skill in the art, can also be used to isolate enantiomerically enriched compounds.

The resulting carboxylic acid-containing compounds can often be easily separated from the amide-containing compounds by simply forming the carboxylate salts, which can be removed in a water extraction and then isolated by acidification of the carboxylate group to reform the carboxylic acid group. The amide groups not hydrolyzed by the enzyme (or, for that matter, via conventional acidic or basic hydrolysis) can be isolated from extraction with an organic solvent, and subjected to alternate hydrolysis conditions.

Optionally, the carboxylic acid groups can be converted to pharmaceutically acceptable carboxylate salts or esters, using known reaction conditions.

5.2.6 Direct Azo Coupling of Nitrobenzenes using Reducing Agents

Nitrobenzene starting materials can be directly coupled to form azo linkages using a reducing agent such as lithium aluminum hydride. Representative reactions using the nitrobenzene compounds are shown below:

In the first instance, the three different azo coupling products will likely have to be separated. In the second instance, the compounds are either identical (X is CO₂H or CO₂ ⁻), or X is readily convertible to a carboxylic acid group, so that separation need not be performed. That is, if X is an aldehyde or primary alcohol, it can be oxidized in the presence of the carboxylic acid group to yield the desired compound. If X is a nitrile or amide group, it can be hydrolyzed in the presence of the carboxylic acid group to yield the desired compound. For these reasons, it may be advantageous to use this embodiment (azo coupling of nitrobenzenes) only when the molecules are either symmetrical or would be symmetrical when X is converted to a carboxylic acid group. It is also worth noting that aldehyde, amide and nitrile groups can be reduced with the reducing agent (for example, lithium aluminum hydride). The stoichiometric equivalents of the reducing agent may need to be adjusted to account for this, and any reduced groups will need to be oxidized to form the carboxylic acid. For this reason, it may be preferred to use compounds where X is a carboxylic acid or carboxylate group when performing this reaction. Finally, any suitable carboxylate salt can be used (that is, any metal cation or ammonium cation can be used) if the desired end product includes a carboxylic acid at that position, as the acidification of virtually any carboxylate salt to form the carboxylic acid is routine in the art. That is to say, for cost reasons, it may be preferred to use a sodium or potassium salt, although other salts would certainly be acceptable.

5.3 Pharmaceutical Formulations

The compounds prepared according to the methods described herein can be included in pharmaceutical formulations, both for veterinary and for human medical use. The compounds can be administered per se as well as in the form of pharmaceutically acceptable esters, salts, and other physiologically functional derivatives thereof.

The formulations include those suitable for parenteral as well as non-parenteral administration, and specific administration modalities include, but are not limited to, oral, rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous, transdermal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial, lymphatic, vaginal, and intra-uterine administration. Formulations suitable for oral and parenteral administration are preferred, with formulations suitable for oral administration most preferred.

In addition to the aforementioned ingredients, the formulations of this invention can further include one or more accessory ingredient(s) selected from diluents, buffers, flavoring agents, disintegrants, surface active agents, thickeners, lubricants, preservatives (including antioxidants), and the like.

The active pharmaceutical ingredient can further include one or more other medicaments including, but not limited to, anti-inflammatory agents such as mesalamine, sulfasalazine, balsalazide, and olsalazine; immunomodulators such as azathioprine, 6-mercaptopurine, cyclosporine and methotrexate; steroidal compounds such as corticosteroids; and antibiotics such as metronidazole and ciprofloxacin. The active pharmaceutical ingredient preferably further comprises mesalamine, the compound of Formula II:

In therapeutic usage, the compounds can be used to treat an animal subject having or latently susceptible to an intestinal condition(s) or disease state(s) and in need of treatment therefor, by administering an effective amount of the compound to the animal. The animal can be a human or non-human animal (e.g., bird, dog, cat, cow, horse), preferably a mammal, and most preferably a human.

The compounds can be used to treat and/or prevent various diseases, particularly inflammatory conditions of the GI tract including, but not limited to, inflammatory conditions of the mouth such as mucositis, infectious diseases (e.g., viral, bacterial, and fungal diseases), and Crobn's disease; inflammatory conditions of the esophagus such as esophagitis, conditions resulting from chemical injury (e.g., lye ingestion), gastroesophageal reflux disease, bile acid reflux, Barrett's esophagus, Crohn's disease, and esophageal stricture; inflammatory conditions of the stomach such as gastritis (e.g., Helicobacter pylori, acid-peptic disease and atrophic gastritis), peptic ulcer disease, pre-cancerous lesions of the stomach, non-ulcer dyspepsia, and Crohn's disease; inflammatory conditions of the intestine such as inflammatory bowel disease, celiac disease, Crohn's disease, bacterial overgrowth, peptic ulcer disease, and fissures of the intestine; inflammatory conditions of the colon such as Crohn's disease, ulcerative colitis, infectious colitis (e.g., pseudomembranous colitis such as Clostridium difficile colitis, salmonella enteritis, shigella infections, yersiniosis, cryptosporidiosis, microsporidial infections, and viral infections), radiation-induced colitis, colitis in the immunocompromised host (e.g., typhlitis), precancerous conditions of the colon (e.g., dysplasia, inflammatory conditions of the bowel, and colonic polyps), proctitis, inflammation associated with hemorrhoids, proctalgia fugax, and rectal fissures; liver gallbladder and/or bilary tract conditions such as cholangitis, sclerosing cholangitis, primary bilary cirrhosis, and cholecystitis; and intestinal abscess. The compounds can also be used to diagnose constituents, conditions, or disease states in biological systems or specimens, as well as for diagnostic purposes in non-physiological systems. Furthermore, the compounds can be used to treat or prevent condition(s) or disease state(s) in plant systems. By way of example, the compounds or individual active components of the conjugate can have insecticidal, herbicidal, fungicidal, and/or pesticidal efficacy amenable to usage in various plant systems.

Depending on the specific condition or disease state, the active pharmaceutical ingredient can be administered at any suitable therapeutically effective and safe dosage, as can readily be determined within the skill of the art and without undue experimentation. For example, the active pharmaceutical ingredient of the present invention can be administered at a dosage between about 0.1 and 200 mg/kg, preferably between about 1 and 90 mg/kg, and more preferably between about 10 and 80 mg/kg.

In some embodiments, the compounds can break down in the intestinal tract to form the metabolic product of Formula III:

where R¹, R³ and R⁴ are as described above with reference to Formula I, and the metabolic product of Formula IV:

The metabolic product of Formula III can possess anti-inflammatory activity and/or immunoregulatory activity. The metabolic product of Formula IV can possess anti-inflammatory activity, and more particularly can inhibit prostaglandin synthetase I & II.

In other embodiments, the compounds can break down in the intestinal tract to form the metabolic product of Formula m and the metabolic product of Formula V:

where R⁶, R⁷ and R⁸ are as described above with reference to Formula I. The metabolic product of Formula V can possess anti-inflammatory activity and/or immunoregulatory activity. Accordingly, the compounds can provide immunoregulatory activity. The compounds can also inhibit prostaglandin synthetase I and II. The compounds can be used to treat various diseases, particularly ulcerative colitis, Crohn's disease and the like.

The present invention will now be described with reference to the following examples. It should be appreciated that these examples are for the purposes of illustrating aspects of the present invention, and do not limit the scope of the invention as defined by the claims.

6. EXAMPLES 6.1 Example 1 Diazo Coupling of 4-Aminophenethyl Alcohol with Salicylic Acid, followed by Oxidation of the Alcohol to the Corresponding Carboxylic Acid:

6.1.1 Diazo Coupling of 4-Aminophenethyl Alcohol with Salicylic Acid:

4-Aminophenethyl alcohol (24.8 mmol) is suspended in water (75 mL) and concentrated hydrochloric acid (8 mL) is added. The solution is cooled to 0° C. in an ice bath with rapid stirring. Sodium nitrite (26.1 mmol) in water (20 mL) is added dropwise to the 4-aminophenylacetic acid solution with rapid stirring. It may be important to keep the temperature between 0-5° C. at all times, especially during the NaNO₂ addition. The reaction is stirred for an additional period of time, for example, about 20 minutes. In the meantime, salicylic acid, sodium salt (74.4 mmol) is dissolved in an aqueous NaOH solution (113 mmol NaOH in 100 ML H₂O). The solution is vigorously stirred at 17° C. and at pH 13.3. The diazonium salt solution is added dropwise to the salicylic acid solution. It can be important to keep the temperature of the salicylic acid solution between 17-18° C. and the pH between 13.2-13.3 at all times, especially during the diazonium salt addition. The temperature can be regulated by adding ice and the pH regulated by adding NaOH, for example, 8 M NaOH. After the addition is complete, the solution is allowed to warm room temperature and stirred for an additional period of time, for example, about 30 minutes. The reaction mixture is then suction filtered to remove any undissolved particulates or unwanted side products. The filtrate is acidified with aqueous HCl (10 mL conc. HCl in 20 mL H₂O) which produces a dark red precipitate. The precipitate is collected by suction filtration and washed several times with cold H₂O, until the filtrate is clear. The collected solid can be air dried overnight.

6.1.2 Oxidation of the Resulting Alcohol to the Corresponding Carboxylic Acid:

A mixture of alcohol formed above (0.7 mol) and a solution of sodium carbonate is placed in 150 mL of water. A solution of potassium permanganate (0.9 mol) in water is added, with vigorous stirring, for 3-4 hours at a temperature of 4-5° C. The reaction mixture is allowed to warm to room temperature and the precipitated manganese dioxide is removed by suction filtration. The solution is concentrated under reduced pressure, a layer of ether is added, and the aqueous solution is acidified with dilute H₂SO₄. The organic and aqueous phases are separated and the aqueous phase extracted 2-3 times with ether. The ether extracts are combined, dried over MgSO₄, and the solvent removed under reduced pressure. The resulting solid can optionally be recrystallized.

6.2 Example 2 Diazo Coupling of 4-Aminobenzyl Cyanide with Salicylic Acid, followed by Hydrolysis of the Nitrite to the Corresponding Carboxylic Acid:

6.2.1 Diazo Coupling of 4-Aminobenzyl Cyanide with Salicylic Acid:

4-Aminobenzyl cyanide (24.8 mmol) is suspended in water (75 mL) and concentrated hydrochloric acid (8 mL) is added. The solution is cooled to 0° C. in an ice bath with rapid stirring. Sodium nitrite (26.1 mmol) in water (20 mL) is added dropwise to the 4-aminophenylacetic acid solution with rapid stirring. It is preferred to keep the temperature between 0-5° C. at all times, especially during the NaNO₂ addition. The reaction is stirred for an additional 20 minutes. In the meantime, salicylic acid, sodium salt (74.4 mmol) is dissolved in an aqueous NaOH solution (113 mmol NaOH in 100 mL H₂O). The solution is vigorously stirred at 17° C. and at pH 13.3. The diazonium salt solution is added dropwise to the salicylic acid solution. It can be extremely important to keep the temperature of the salicylic acid solution between 17-18° C. and the pH between 13.2-13.3 at all times, especially during the diazonium salt addition. The temperature is regulated by adding ice and the pH regulated by adding NaOH, for example, 8 M NaOH. After the addition is complete, the solution is allowed to warm room temperature and stirred for an additional period of time, such as about 30 minutes. The reaction mixture can be suction filtered to remove any undissolved particulates or unwanted side products, and the filtrate can be acidified with aqueous HCl (10 mL conc. HCl in 20 mL H₂O) to produce a dark red precipitate. The precipitate can be collected by suction filtration and washed several times with cold H₂O, until the filtrate is clear. The collected solid can be air dried overnight.

6.2.2 Hydrolysis of the Nitrile to the Corresponding Carboxylic Acid

a) Basic Conditions:

The nitrile formed above (1.2 mol) and a solution of NaOH (2.3 mol) can be placed in H₂O (260 mL) in a round-bottom flask and heat to reflux using a condenser for approximately 5-10 h. Water (100 mL) can be added through the condenser, then slowly with external cooling, 50% H₂SO₄ can be added. The upper layer containing the desired carboxylic acid can be separated and extracted with CH₂Cl₂. The organic extracts can be combined, dried over MgSO₄, and the solvent removed under reduced pressure. The resulting solid can optionally be recrystallized.

b) Acidic Conditions:

The nitrile formed above (0.85 mol) and a solution of 50:50 H₂SO₄:glacial acetic acid can be placed in H₂O (100 mL) in a round-bottom flask and heated to reflux using a condenser for approximately 1 hour. The reaction mixture can be allowed to cool to room temperature and poured into 2-3 volumes of water with stirring. The desired crude product should precipitate. The resulting solid can be collected by suction filtration and washed with water until the filtrate is neutral. The resulting solid can optionally be recrystallized.

6.3 Example 3 Reduction of Nitro Compounds to form an Azo Linkage

In this embodiment, two nitrobenzene compounds (shown below as ArNO₂) are coupled to form a direct azo linkage between the two compounds. This embodiment will result in mixtures of products (A-A, A-B and B-B) if two different nitrobenzene compounds are used. For this reason, it may be preferably to limit this synthetic approach to the synthesis of symmetrical azo compounds, e.g. 4-APAA dimer, etc. (Ar=Phenyl ring)

A solution of NaOH (1.6 mol) in H₂O (150 mL), nitrobenzene (0.41 mol) and MeOH (500 mL) is placed in a 3-neck flask equipped with an overhead stirrer and reflux condenser. Zn powder (0.9 mol) is added and refluxed with vigorous stirring for approximately 10 h. The reaction mixture is hot filtered and the precipitate washed with MeOH. Concentrated HCl is added to the filtrate until the pH is neutral by litmus paper, and the precipitated is re-filtered. The MeOH is removed under reduced pressure and the resulting aqueous solution is cooled in an ice bath until the desired azo compound precipitates. The solid can then be collected by suction filtration and optionally recrystallized.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. 

1. A method for preparing a compound of the formula:

where R¹, R³ and R⁴ are is selected from the group consisting of H, CH₃, CH₂CH₃, CH(CH₃)₂, and C₁-C₄ alkyl; and R² is:

where R⁵, R⁶, R⁷ and R⁸ are independently hydrogen or C₁ to C₄ alkyl; or the esters or pharmaceutically acceptable salts thereof, comprising: (a) converting a compound of one of the following formulas:

to a diazonium salt; (b) reacting the diazonium salt with a compound of one of the following formulas:

(c) oxidizing the primary alcohol or aldehyde group to a carboxylic acid group, and (d) optionally forming an ester or pharmaceutically acceptable salt of one or more of the carboxylic acid groups.
 2. The method of claim 1, wherein R¹, R³, and R⁴ are independently selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂.
 3. The method of claim 1, wherein R⁶, R⁷, and R⁸ are independently selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂.
 4. The method of claim 1, wherein R⁵ is selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂.
 5. The method of claim 1, wherein the compound that is formed is 5-(4-carboxymethyl-phenylazo)-2-hydroxy-benzoic acid.
 6. The method of claim 1, wherein the compound that is formed is 5-[4-1-carboxy-ethyl)-phenylazo]-2-hydroxy-benzoic acid.
 7. The method of claim 1, wherein the compound that is formed is 4-(4-carboxymethyl-phenylazo)-phenylacetic acid.
 8. The method of claim 1, wherein the aniline starting material is formed by reducing a nitrobenzene of one of the following formulas:


9. The method of claim 1, wherein the oxidation of the primary alcohol or aldehyde group is performed using a chromium (VI) oxidizing agent.
 10. The method of claim 9, wherein the chromium (VI) oxidizing agent is present in catalytic amounts, and a co-oxidant is used to regenerate the chromium (VI) oxidizing agent.
 11. The method of claim 1, wherein the compound exists as a mixture of diastereomers, further comprising isolating an individual enantiomer from the mixture of diastereomers.
 12. A method for preparing a compound of the formula:

where R¹, R³ and R⁴ are is selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂; and R² is:

where R⁵, R⁶, R⁷ and R⁸ are independently hydrogen or C₁ to C₄ alkyl; or the esters or pharmaceutically acceptable salts thereof, comprising: (a) converting a compound of one of the following formulas:

to a diazonium salt; (b) reacting the diazonium salt with a compound of one of the following formulas:

(c) hydrolyzing the nitrile or amide group to form a carboxylic acid group, and (d) optionally forming an ester or pharmaceutically acceptable salt of one or more of the carboxylic acid groups.
 13. The method of claim 12, wherein R¹, R³, and R⁴ are independently selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂.
 14. The method of claim 12, wherein R⁶, R⁷, and R⁸ are independently selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂.
 15. The method of claim 12, wherein R⁵ is selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂.
 16. The method of claim 12, wherein the compound that is formed is 5-(4-carboxymethyl-phenylazo)-2-hydroxy-benzoic acid.
 17. The method of claim 12, wherein the compound that is formed is 5-[4-(1-carboxy-ethyl)-phenylazo]-2-hydroxy-benzoic acid.
 18. The method of claim 12, wherein the compound that is formed is 4-(4-carboxymethyl-phenylazo)-phenylacetic acid.
 19. The method of claim 12, wherein the aniline starting material is formed by reducing a nitrobenzene of one of the following formulas:


20. The method of claim 12, wherein the nitrile or amide is hydrolyzed to the carboxylic acid using acid catalysis.
 21. The method of claim 12, wherein the nitrile or amide is hydrolyzed to the carboxylic acid using basic catalysis.
 22. The method of claim 12, wherein the nitrile or amide is hydrolyzed to the carboxylic acid using enzymatic catalysis.
 23. The method of claim 22, wherein the isolation step involves enzymatic resultion of one of the enantiomers during the hydrolysis of a nitrile or amide group.
 24. A method for preparing a compound of the formula:

where R¹, R³ and R⁴ are is selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂; and R² is:

where R⁵, R⁶, R⁷ and R⁸ are independently hydrogen or C₁ to C₄ alkyl; or the esters or pharmaceutically acceptable salts thereof, comprising: a) combining a first compound of formula

where X is selected from the group consisting of CH₂OH, C(O)H, CO₂H, CO₂ ⁻, CN, and C(O)NR¹ ₂, with a second compound of formula

b) adding a reducing agent that is capable of reducing the nitro groups in the first and second compounds and forming an azo group, and c) converting any X groups that are not in the form of carboxylic acid, carboxylate salt or ester groups to such groups.
 25. The method of claim 24, wherein R¹, R³, and R⁴ are independently selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂.
 26. The method of claim 24, wherein R⁶, R⁷, and R⁸ are independently selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)2.
 27. The method of claim 24, wherein R⁵ is selected from the group consisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂.
 28. The method of claim 24, wherein the compound that is formed is 5-(4-carboxymethyl-phenylazo)-2-hydroxy-benzoic acid.
 29. The method of claim 24, wherein the compound that is formed is 5-[4-(1-carboxy-ethyl)phenylazo]-2-hydroxy-benzoic acid.
 30. The method of claim 24, wherein the compound that is formed is 4-(4-carboxymethyl-phenylazo)-phenylacetic acid.
 31. The method of claim 24, further comprising removing products formed by the coupling of two of the first compounds and/or two of the second compounds from the desired product resulting from the coupling of the first and second compound.
 32. The method of claim 24, wherein X is CN or C(O)NR¹ ₂, wherein the CN or C(O)NR¹ ₂ groups are either hydrolyzed to form carboxylic acids or carboxylate salts, or, in the event that the CN or C(O)NR¹ ₂ groups are reduced by the reducing agent during the coupling of the nitro groups, such reduced groups are converted to carboxylic acid groups or carboxylate salts.
 33. The method of claim 32, wherein one or more of the carboxylic acids or carboxylate salts are esterified.
 34. The method of claim 24, wherein X is CH₂OH or C(O)H, further comprising oxidizing the CH₂OH or C(O)H groups to form a carboxylic acid.
 35. The method of claim 24, wherein the oxidation of the primary alcohol or aldehyde group is performed using a chromium (VI) oxidizing agent.
 36. The method of claim 35, wherein the chromium (VI) oxidizing agent is present in catalytic amounts, and a co-oxidant is used to regenerate the chromium (VI) oxidizing agent.
 37. The method of claim 24, wherein the compound exists as a mixture of diastereomers, further comprising isolating an individual enantiomer from the mixture of diastereomers. 